基于TCID50检测AAV9载体制品感染性滴度的方法

目的:建立一种基于半数组织培养感染剂量(median tissue culture infective dose,TCID₅₀)检测9型腺相关病毒(adeno-associated virus type 9,AAV9)载体制品感染性滴度的方法。方法:利用含AAV2 rep和cap基因的1型单纯疱疹病毒(herpes simplex virus type1,HSV1)做为辅助病毒与梯度稀释的AAV9载体制品共同感染HEK-293细胞,培养48 h后用实时荧光定量PCR(quantitative real-time PCR,qPCR)扩增AAV特异性反向末端重复序列(inverted terminal repeats,ITR),根据阳性及阴性感染孔数,利用Kärber法计算样品的TCID₅₀。结果:采用携带增强绿色荧光蛋白报告基因的AAV9载体制品确定辅助病毒HSV1-rc最佳感染复数(multiplicity of infection,MOI)为5,AAV9-101的感染性滴度为1.6×10⁹ TCID₅₀/mL。结论:对AAV9载体制品进行感染性滴度检测,且具有可重复性。

Research on AAV9 Infectious Titer Detection Method Based on TCID50

Objective: To establish a method for detecting the infectious titers of adeno-associated virus type 9 (AAV9) vector products based on the median tissue culture infective dose (TCID₅₀). Methods: HEK-293 cells were co-infected with HSV1 containing rep and cap genes of AAV2 and gradient diluted AAV9 vector products. After 48 hours of culture, the AAV-specific inverted terminal repeats (ITR) were amplified by quantitative real-time PCR(qPCR), and the infectivity titer of the sample was calculated by the Kärber method based on the number of positive and negative infected wells. Results: The AAV9 vector product carrying the enhanced green fluorescent protein reporter gene was used to determine the optimal multiplicity of infection (MOI) of the helper virus HSV1-rc as 5. The infectious titer of AAV9-101 is 1.6×10⁹ TCID₅₀/mL. Conclusion: This method can detect the infectious titer of AAV9 vector products with repeatability.